Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2^−/− embryonic stem cells failed to maintain γ-irradiation–induced arrest in the G₂ phase of the cell cycle. Chk2^−/−thymocytes were resistant to DNA damage–induced apoptosis. Chk2^−/− cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to γ irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to γ irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.